Methods and materials for modulating resistance to apoptosis

ABSTRACT

This document provides methods and materials involved in modulating a cell&#39;s ability to be resistant to apoptosis. For example, methods and materials for exposing cells to KLK6 polypeptides, or increased KLK6 polypeptide activity, to promote resistance to apoptosis are provided. In addition, methods and materials for reducing the ability of KLK6 polypeptides to promote resistance to apoptosis are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/388,289, filed Sep. 30, 2010.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersNS052741 and RG3367 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in modulating acell's ability to be resistant to apoptosis. For example, this documentrelates to methods and materials for using, for example, kallikrein 6polypeptides to promote resistance to apoptosis. This document alsorelates to methods and materials for using, for example, kallikrein 6polypeptide inhibitors to reduce resistance to apoptosis.

2. Background Information

Kallikrein 6 (KLK6) polypeptides are members of the kallikrein genefamily that includes secreted serine proteases aligned on humanchromosome 19q13.3-4. These gene family members form the largestcontiguous cluster of serine proteases in the human genome.

SUMMARY

This document provides methods and materials involved in modulating acell's ability to be resistant to apoptosis. For example, this documentprovides methods and materials for exposing cells to KLK6 polypeptides,or increased KLK6 polypeptide activity, to promote resistance toapoptosis. As described herein, the exposure of wide range of differentcells to KLK6 polypeptides promotes the survival of those cells underresting conditions and/or under conditions that normally promoteapoptosis. For example, lymphocytes, monocytes, oligodendrocytes,astrocytes, and glioma cells normally undergo measurable apoptosis upontreatment with an apoptosis-inducing agent (e.g., camptothecin, ConA, orstaurosporine). Treatment with KLK6 polypeptides, however, promotes cellsurvival of cells exposed to an apoptosis-inducing agent, therebyreducing the apoptosis-inducing effects of the apoptosis-inducing agent.As described herein, KLK6 polypeptides, molecules designed to increaseKLK6 polypeptide expression levels, and molecules designed to increaseKLK6 polypeptide activity can be used to treat conditions that exhibitundesirable apoptosis. For example, conditions and diseases that involvetoo much cell death can be treated using KLK6 polypeptides, moleculesdesigned to increase KLK6 polypeptide expression levels, or moleculesdesigned to increase KLK6 polypeptide activity. Having the ability toincrease survival of cells that may undergo apoptosis can allowclinicians and other health care professionals to reduce the effects andsymptoms associated with, for example, excessive apoptosis.

This document also provides methods and materials for reducing theability of KLK6 polypeptides to promote resistance to apoptosis. Forexample, an inhibitor of KLK6 polypeptide activity can be used to reduceKLK6 polypeptide-induced resistance to apoptosis. As described herein,KLK6 polypeptide inhibitors and molecules designed to reduce KLK6polypeptide expression can be used to treat conditions and diseases thatexhibit undesirable resistance to apoptosis. Having the ability toreduce the resistance to apoptosis can allow clinicians and other healthcare professionals to reduce the effects and symptoms associated with,for example, cells exhibiting excessive resistance to apoptosis.

One advantage of using KLK6 polypeptides as a therapeutic target toregulate cell survival/death is that this polypeptide is a secretedenzyme that acts in the extracellular space, thereby eliminating theneed for intracellular targeting strategies. Another advantage of usingKLK6 polypeptides as a therapeutic target is that KLK6 polypeptidesappear to have broad physiological relevance to a wide range of celltypes and clinical disorders.

In general, one aspect of this document features a method for treating amammal having a condition wherein cells undergo excessive apoptosis. Themethod comprises, or consists essentially of, administering acomposition comprising, or consists essentially of, a KLK6 polypeptideor a nucleic acid encoding said KLK6 polypeptide to the mammal underconditions wherein the composition reduces the level of apoptosis of thecells. The composition can comprise, or consist essentially of, the KLK6polypeptide. The composition can comprise, or consist essentially of,the nucleic acid encoding the KLK6 polypeptide. The mammal can be ahuman. The condition can be a condition resulting from the withdrawal ofgrowth factors or the activation of cell surface death receptors. Thecondition can be a condition resulting from exposure to heat shock,hypoxia, UV radiation, dexamethasone, cytotoxic agents, orchemotherapeutic agents.

In another aspect, this document features a method for treating a mammalhaving cancer. The method comprises, or consists essentially of,administering a composition comprising, or consisting essentially of, aninhibitor of a KLK6 polypeptide expression or activity to the mammalunder conditions wherein the composition increases the level ofapoptosis of cancer cells within the mammal. The composition cancomprise, or consist essentially of, a KLK6 antisense molecule or a KLK6miRNA molecule. The composition can comprise, or consist essentially of,an anti-KLK6 polypeptide antibody. The mammal can be a human.

In another aspect, this document features a method for treating a mammalhaving an inflammatory condition. The method comprises, or consistsessentially of, administering a composition comprising, or consistingessentially of, an inhibitor of a KLK6 polypeptide expression oractivity to the mammal under conditions wherein the compositionincreases the level of apoptosis of cells within the mammal. In somecases, the method can include monitoring the level of apoptosis withinthe mammal. The composition can comprise, or consist essentially of, aKLK6 antisense molecule or a KLK6 miRNA molecule. The composition cancomprise, or consist essentially of, an anti-KLK6 polypeptide antibody.The mammal can be a human.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-D contain graphs demonstrating the differential effects of KLK1and KLK6 polypeptides on survival and proliferation of murinesplenocytes. Murine splenocytes were labeled with CFSE(carboxyfluorescein succinimidyl ester) and cultured in defined media inthe presence of 1 or 10 μg/mL of KLK1 or KLK6 polypeptides, or vehiclealone (control), for periods of 24 or 72 hours. At harvest, dead cellswere labeled using PI (propidium iodide), and samples were examined byflow cytometry. FIG. 1A contains graphs demonstrating that stimulationof cultures with either 1 or 10 μg/mL of KLK6 polypeptides, but not KLK1polypeptides, significantly reduced the number of dead (PI+) cellsobserved at either the 24- or 72-hour time points. FIG. 1B is a graphplotting the results obtained as 72 hours. FIG. 1C contains graphsplotting the intensity of CFSE labeling in PI− cells determined usingthe proliferation platform of the Flow Jo Program and labeling peaksobserved after 24 hours. FIG. 1D contains a graph demonstrating thatKLK1 polypeptides (10 μg/ml), but not KLK6 polypeptides, promoted asignificant increase in the percent of cells divided at the 24-hour timepoint. Data are expressed as mean+SEM, One Way ANOVA with SNK post hoctest; P<0.001**, P<0.02*; (SSC, side scatter).

FIG. 2 contains graphs demonstrating that KLK6 blocks cisplatin inducedlymphocyte death. Recombinant KLK6 blocks death of murine T and B cellsunder resting conditions and in the presence of 25 μg/mL of thechemotherapeutic cisplatin (*P<0.05, Students t-test). Percent celldeath was determined by quantification of propidium iodide labeled cellsusing flow cytometry. Results from 24 hour exposure times are shown.Panel (A) contains a graph plotting results for total cell death. Panel(B) contains a graph plotting results for T cell death. Panel (C)contains a graph plotting results for B cell death.

FIG. 3 contains graphs demonstrating that KLK6 blocks ATP inducedlymphocyte death. Recombinant KLK6 blocks death of murine T and B cellsunder resting conditions and in the presence of 100 μM ATP, which isknown to trigger calcium influx and apoptosis (*P<0.05, Studentst-test). Percent cell death was determined by quantification ofpropidium iodide labeled cells using flow cytometry. Results from 24hour exposure times are shown. Panel (A) contains a graph plottingresults for total cell death. Panel (B) contains a graph plottingresults for T cell death. Panel (C) contains a graph plotting resultsfor B cell death.

FIGS. 4A-D contain graphs demonstrating that KLK6 polypeptides blockdeath of murine splenocytes and Jurkat T cells across multipleparadigms. KLK6 (10 μg/mL) significantly reduced the number of PI+ deadcells observed by flow cytometry after a 24-hour period in culture underresting conditions (A), in response to (0.1 μM) dexamethasone, or a24-hour exposure to increasing concentrations of Fas ligand (FasL, B).In parallel experiments, Jurkat T cells exhibited significantly reducedlevels of cell death under resting conditions and in response tocamptothecin (1.0 μM) or Fas ligand (D, 2 μg/mL). KLK6 polypeptidesblocked death of both CD3+ T cells and B220+ B cells under restingconditions (C). KLK6 polypeptides were applied at the time of plating inconjunction with cell death inducing agents in each case. A small butsignificant improvement in the ability of KLK6 polypeptides to rescuesplenocytes from death was seen with a 2-hour pre-incubation prior tothe addition of dexamethasone (A). Data are expressed as mean+SEM, OneWay ANOVA with SNK post hoc test for multiple comparisons in FIG. 1A,two-way comparisons FIGS. 1A to D were made using Students t-test;P<0.001***, P<0.005**, P<0.02*.

FIGS. 5A-C contain graphs demonstrating that as little as a 5-minutepulse of KLK6 polypeptides is sufficient to promote splenocyte survival.To gauge the minimal time of exposure to KLK6 polypeptides necessary toobserve its pro-survival effects, splenocytes were pulsed with KLK6 (10μg/mL) for periods of 5, 30, or 60 minutes, or in the presence of KLK6polypeptides for the full 24- or 48-hour period of culture examinedprior to labeling with PI for analysis of dead cells by flow cytometry(FIGS. 5A and B). A 5-minute pulse with KLK6 polypeptides promotedsignificant survival of explanted splenocytes over a 24-hour cultureperiod that was similar in magnitude to that seen with longer pulses,i.e., 30 or 60 minutes and 24 hours (FIG. 5A). A 5-minute pulse withKLK6 polypeptides was also sufficient to promote survival when cellswere exposed to dexamethasone (0.1 μM) for 24 hours (FIG. 5C). Afterlonger periods of culture, however (FIG. 5B, 48 hours), only the moreprolonged period of KLK6 polypeptide stimulation (48 hours) exertedsignificant pro-survival effects (FIG. 5B). Data are expressed asmean+SEM, One Way ANOVA with SNK post hoc test for multiple comparisons;P<0.001**, P<0.005*.

FIGS. 6A and B provide results demonstrating that the ability of KLK6polypeptides to promote survival of murine splenocytes relates to itsability to block the apoptotic cascade. FIG. 6A is a graph demonstratingthat KLK6 polypeptides significantly reduced the number of dead cells(Annexin V+ and PI+) observed in cultures of splenocytes under restingconditions and after exposure to dexamethasone (0.1 μM) or staurosporine(1 μM). Reduced cell death was accompanied by a significant increase inthe live population (unlabeled cells) in the case of cells under restingconditions and those exposed to staurosporine. KLK6 polypeptides alsopromoted an accumulation of splenocytes at early apoptotic stages(Annexin V+, PI−) in the presence of apoptosis inducing agents,suggesting KLK6 polypeptides delay the apoptotic cascade. Data areexpressed as mean+SEM; One Way ANOVA with SNK post hoc test for multiplecomparisons, P<0.001***, P<0.003**, P<0.007*. FIG. 6B includes aphotograph of a Western blot demonstrating that KLK6 polypeptides (10μg/mL)-induced reductions in the amount of cleaved PARP observed inmurine splenocytes cultured for a 24-hour period under restingconditions or in the presence of camptothecin (1.0 μM). The blot wasre-probed for Actin to confirm loading accuracy.

FIGS. 7A-D contain graphs demonstrating that the ability of KLK6polypeptides to rescue murine T and B cells from death depends in parton activation of PAR1. To determine the involvement of PAR1 inKLK6-mediated lymphocyte rescue, the effects of KLK6 polypeptides (10μg/mL) were compared between splenocytes harvested from wild type orPAR1−/− mice. FIG. 7A is a graph demonstrating that the absence of PAR1blocked the ability of KLK6 polypeptides to rescue CD3+ T cells fromdeath under resting conditions but in the presence of dexamethasone (0.1μM) any effect of PAR1 deletion did not reach the level of statisticalsignificance. FIG. 7B is a graph demonstrating that PAR1 deficiency alsoblocked the ability of KLK6 polypeptides to rescue B220+ B cells fromresting cell death and significantly reduced KLK6 polypeptide-mediated Bcell rescue in the presence of dexamethasone. FIG. 7C is a graphdemonstrating that neither PAR1- or PAR2-APs alone, or in combinationwith PAR4-AP (FIG. 7D), were able to mimic the pro-survival effects ofKLK6 polypeptides. PAR1-AP alone in the presence of dexamethasoneexacerbated cell death. Data are expressed as mean+SEM; two-waycomparisons A and B, made using Students t-test, P<0.003***, P<0.009**,P=0.03; One Way ANOVA with SNK post hoc test for multiple comparisons inFIGS. 7C and D, P<0.001***, P<0.002**; P<0.012*.

FIG. 8 is a bar graph plotting total cell death results that demonstratethat the ability of KLK6 to block lymphocyte cell death is reduced inthe absence of the G-protein coupled receptor protease activatedreceptor 2 (PAR2). Recombinant KLK6 blocks death of murine lymphocytesunder resting conditions and in the presence of 1 μg/mL dexamethasone.The extent of KLK6-mediated rescue is significantly reduced inlymphocytes derived from PAR2 deficient (−/−) mice (*P<0.05, Studentst-test). Percent cell death was determined by quantification ofpropidium iodide labeled cells using flow cytometry. Results from 24hour exposure times are shown.

FIGS. 9A and B contain photographs of Western blot results demonstratingthat KLK6 polypeptides differentially regulate Bcl2 family members in aPAR1 dependent fashion. In particular, the Western blots show the effectof KLK6 polypeptides on Bcl-XL and Bim in splenocytes derived from wildtype and PAR1 deficient mice. KLK6 polypeptides promoted the expressionof the pro-survival protein Bcl-XL in the presence of camptothecin orConA (FIG. 9A), and this effect was abolished in the absence of PAR1(FIG. 9B). KLK6 polypeptides also suppressed the expression of thepro-apoptotic protein Bim in all conditions examined (FIG. 9A), and thiseffect was reduced or absent in PAR1 deficient mice (FIG. 9B). Actin wasused to control for loading in each case.

FIG. 10 is a graph plotting percent cell death of murine T cells (CD3+),monocytes (CD11b+), or B cells (B220+) treated with or without 10 μg/mLof recombinant KLK6 polypeptides. *=P<0.05 (One Way ANOVA, SNK post hoctest).

FIGS. 11A-D contain graphs demonstrating that KLK6 polypeptides promotessurvival of Jurkat T cells in part by slowing the apoptotic cascade. Toaddress the effect of KLK6 polypeptides on apoptosis, Jurkat cells weregrown under resting conditions (FIG. 11A), or in the presence ofapoptosis inducing agents (FIGS. 11B to D), then labeled with AnnexinV-PE and 7-AAD prior to flow cytometry. Under resting conditions (FIG.11A), and in the presence of camptothecin (FIG. 11B, 1.0 μM), ConA (FIG.11C, 5 μg/mL), or ConA plus camptothecin (FIG. 11D), KLK6 polypeptides(10 μg/mL) promoted an increase in the number of live cells (unlabeled)and a decrease in the number of dead cells (Annexin V+ and 7AAD+) atboth acute (4 hours), subacute (24 hours) and in some cases the morechronic time point examined (48 hours). In the presence of apoptosisinducing agents (FIGS. 11B to D), KLK6 polypeptides promoted asignificant increase in the number of Jurkat cells positive for AnnexinV, but negative for 7AAD, and therefore classified as in the earlystages of apoptosis. Data are expressed as mean+SEM, One Way ANOVA withSNK post hoc test for multiple comparisons; P<0.001***, P<0.005**,P<0.02*.

FIG. 12 contains a graph (A) and flow cytometry results (B)demonstrating that KLK6 over expression in Jurkat leukemia T cellsreduces cell death. Jurkat T cells were stably transduced with a vectorin which the human KLK6 gene is constitutively expressed under thecontrol of a CMV promoter, or with an empty vector (Control), and levelsof live (Annexin V-PE− and 7AAD−), early apoptotic (Annexin V-PE+ and7AAD−), or dead (Annexin V-PE+ and 7AAD+) cells were determined by flowcytometry under resting conditions, or after 24 hour periods of exposureto 0.1 or 0.25 μM staurosporine. (A) Histogram and corresponding dotplots (B), demonstrate KLK6 over expression produces effects largelyparallel to those afforded by treatment of cultures with recombinantKLK6. There was a decrease in the percentage of dead cells and anincrease in the number of live cells in Jurkat T cells over expressingKLK6, relative to those expressing an empty vector. KLK6 over expressionalso reduced cell death in the presence of 0.1 or 0.25 μM staurosporinerelative to that seen in cells stably transduced with empty vector.Reductions in cell death were likely to reflect in part a delay inapoptosis, since after 24 hour exposure to 0.25 μM Staurosporine, KLK6over expression not only reduced the number of dead cells and increasedthe number of live cells, but cells in the early stages of apoptosis(AnnV-PE+, 7AAD−) were also significantly elevated. Data are expressedas mean+SEM of triplicate cultures examined in parallel; One Way ANOVAwith SNK post hoc test for multiple comparisons, P<0.001***, P=0.002**,P=0.003*. All data shown is representative of that seen in at leastthree independent cell culture experiments.

FIG. 13 is a graph plotting the percent of oligodendrocyte cell deathfor cells exposed to staurosporine (0.5 μM) in the absence or presenceof recombinant KLK6 polypeptides (1, 5, or 10 μg/mL). *=P<0.001 (One WayANOVA, SNK post hoc test).

FIG. 14 is a graph plotting the percent of human glioma cell death forcells exposed to staurosporine (1 μM) in the absence or presence(pre-exposed to KLK6 polypeptides for 2 hour prior to application ofstaurosporine) of recombinant KLK6 polypeptides (10 μg/mL). *=P<0.005(One Way ANOVA, SNK post hoc test).

FIG. 15 contains a graph (A) and flow cytometry results (B)demonstrating that recombinant KLK6 promotes the resistance of the U251GBM cell line to the pan-protein kinase inhibitor Staurosporine in adose dependent fashion (*P<0.05, Students t-test). The percent of cellslabeled with propidium iodide (PI) after 24 hour exposure tostaurosporine (1μM) shown in histogram (A) was quantified by flowcytometry (B) and is taken as a measure of cell death. All samples weretreated and analyzed in triplicate. Both 5 and 10 μg/mL recombinant KLK6significantly reduced the amount of staurosporine induced cell death.

FIG. 16 contains a graph (A) and flow cytometry results (B)demonstrating that recombinant KLK6 significantly reducesstaurosporine-induced apoptosis of the U251 GBM line. Levels of live(Annexin V - and Propidium iodide- (PI−)), early apoptotic (Annexin V+and PI−), or dead (Annexin V+ and PI+) cells were determined by flowcytometry after a 24 hour period of exposure to 1 μM staurosporine (B)or staurosporine in the presence of 10 μg/mL KLK6. Histogram (A) showsquantification of three separate samples for each condition. KLK6significantly increased the number of live cells and reduced the numberof early apoptotic and dead cells (*P<0.05, Students t-test).

FIG. 17 contains results demonstrating that KLK6 over expressionpromotes resistance of the U251 GBM cell line to staurosporine induceddeath. (A) Shows flow cytometry dot blots of the U251 GBM cell linetransfected with a control CMV driven construct alone or a vectorcontaining a KLK6-CMV driven construct. Dead cells were detected bylabeling with 7-AAD. Panel (B) is a histogram showing a quantitativeanalysis of triplicate samples demonstrating cells transfected with theKLK6 expressing vector (KLK6-CMV) exhibited significantly reduced celldeath in the presence of 1 μM staurosporine. Panel (C) containsquantitative real time PCR results showing that cells transfected withthe KLK6-CMV construct express significantly higher levels of KLK6 mRNA.All expression data were normalized to the constitutively expressed geneGAPDH (*P<0.05, Students t-test).

FIG. 18 contains results demonstrating that U251 GBM cells overexpressing KLK6 were associated with increased resistance to treatmentwith radiation (RT) or temozolomide (TMZ). Control U251 GBM cells orU251 cells transfected with an empty control-CMV vector, or with aKLK6-CMV vector were plated at low density, treated with 2 or 5 Gy ofionizing radiation, or with 10 μM TMZ for 24 hours and then allowed toproliferate for 2 weeks in culture. Cellular colonies formed were thenfixed with acidic crystal violet (B), and the number of colonies formedwas quantified as shown in histogram (A). U251 cells over expressingKLK6 (KLK6-CMV) were significantly more resistant to either 2 or 5 Gyradiation, or TMZ, as reflected in an increased number of coloniescounted (*P<0.05, Students t-test).

FIG. 19 contains results demonstrating that U251 GBM cells overexpressing KLK6 were resistant to combined treatment with radiation (RT)and temozolomide (TMZ), the current standard of care for patients withGBM. Control U251 GBM cells, or U251 cells transfected with an emptycontrol-CMV vector, or with a KLK6-CMV vector were plated at lowdensity, treated with 2 Gy of ionizing radiation and 10 μM TMZ (24hours), and then allowed to proliferate for 2 weeks in culture. Coloniesformed were then fixed with acidic crystal violet (B), and the number ofcolonies formed was quantified as shown in histogram (A). U251 cellsover expressing KLK6 (KLK6-CMV) were significantly more resistant tocombined treatment with RT and TMZ as reflected in an increased numberof colonies counted (*P<0.05, Students t-test).

FIG. 20 contains results demonstrating that U251 GBM cells overexpressing KLK6 were associated with increased resistance to treatmentwith the chemotherapeutic agent, Cisplatin. Control U251 GBM cells orU251 cells transfected with an empty control-CMV vector, or with aKLK6-CMV vector were plated at low density, treated with 10 μg/mL ofCisplatin for 24 hours, and then allowed to proliferate for 2 weeks inculture. Colonies formed were then fixed with acidic crystal violet (A),and the number of colonies formed quantified. Significantly morecolonies were seen in GBM cells over expressing KLK6. (B) Cell death inresponse to 24 hour exposure to Cisplatin (2.5 μg/mL) was also measuredby flow cytometry using PI to label dead cells. As revealed by theclonogenicity assay, U251 GBM cells over expressing KLK6 (KLK6-CMV) weremore resistant to 24 hour exposure to Cisplatin than were cellsexpressing an empty control vector. Histogram represents triplicatesamples assessed by flow cytometry (*P<0.05, Students t-test).

FIG. 21 contains results demonstrating that knock down of PARI in U251GBM cells diminishes the ability of KLK6 to promote resistance tostaurosporine induced cell death. U251 GBM cells were stably transfectedwith a vector containing a PARI-specific small interfering RNA expressedfrom a short hairpin RNA (shPAR1) or with a no template control vector(shNT). Recombinant KLK6 was not able to significantly reducestaurosporine induced apoptosis in shPAR1 expressing U251 cells,although significant rescue was preserved in those cells expressing theno template control vector alone (*P<0.05, Students t-test).

FIG. 22 contains a listing of a nucleic acid sequence (A) and an aminoacid sequence (B) of a human KLK6 polypeptide. The amino acid sequenceof a mature active KLK6 polypeptide is underlined.

DETAILED DESCRIPTION

This document provides methods and materials involved in modulating acell's ability to be resistant to apoptosis. As described herein, KLK6polypeptide activity can either be increased to reduce apoptosis, orinhibited to promote apoptosis. For example, this document providesmethods and materials for exposing cells to kallikrein 6 (KLK6)polypeptides, or increased KLK6 polypeptide activity, to promoteresistance to apoptosis. As described herein, the exposure of cells toKLK6 polypeptides promotes the survival of those cells under restingconditions and/or under conditions that normally promote apoptosis.Examples of cells that can be exposed to a KLK6 polypeptide (orincreased KLK6 polypeptide activity) include, without limitation,lymphocytes (e.g., T cells or B cells), monocytes, oligodendrocytes,astrocytes, glioma cells, epidermal cells, or stem cells. Anyappropriate mammal can be treated using the methods and materialsdescribed herein. For example, mammals such as humans, monkeys, dogs,cats, cows, horses, pigs, rats, and mice can be treated as describedherein.

As described herein, KLK6 polypeptides, molecules designed to increaseKLK6 polypeptide expression levels, and molecules designed to increaseKLK6 polypeptide activity can be used to treat conditions that exhibitundesirable apoptosis. For example, conditions and diseases that involvetoo much cell death can be treated using KLK6 polypeptides, moleculesdesigned to increase KLK6 polypeptide expression, or molecules designedto increase KLK6 polypeptide activity. Examples of such conditions anddiseases include, without limitation, immunodeficiency disorders (e.g.,immunodeficiency disorders induced by HIV infections), anemia,lymphyopenia, and sepsis. In some cases, an abnormal loss of neurons andglia associated with a neurodegenerative disease such as Huntington'sdisease, Parkinson's disease, Alzheimer's disease, and retinal/maculardegeneration can be reduced by administering KLK6 polypeptides,molecules designed to increase KLK6 polypeptide expression, and/ormolecules designed to increase KLK6 polypeptide activity. In some cases,excessive apoptosis can occur during heart failure, stroke, liverinjury, kidney disease, multiple organ dysfunction syndrome, and bonedisorders. In such cases, KLK6 polypeptides, molecules designed toincrease KLK6 polypeptide expression, and/or molecules designed toincrease KLK6 polypeptide activity can be administered to reduce celldeath, thereby reducing the severity of one or more symptoms of theseconditions.

In some cases, KLK6 polypeptides, molecules designed to increase KLK6polypeptide expression, and/or molecules designed to increase KLK6polypeptide activity can be used to promote resistance to cell deaththat occurs with ischemic injury to tissues including, withoutlimitation, muscle, heart, brain, and gastrointestinal tissue. In somecases, KLK6 polypeptides, molecules designed to increase KLK6polypeptide expression, and/or molecules designed to increase KLK6polypeptide activity can be used to reduce apoptosis and promote cellsurvival to enhance tissue regeneration, to improve responses toimmunological adjuvants, and to enhance stem cell therapy.

In some cases, KLK6 polypeptides, molecules designed to increase KLK6polypeptide expression, and/or molecules designed to increase KLK6polypeptide activity can be used to reduce excessive apoptosis that canoccur in trophoblast cells of the placenta as they invade the uterineenvironment, thereby preventing complications of pregnancy such aspreeclampsia. In some cases, KLK6 polypeptides, molecules designed toincrease KLK6 polypeptide expression, and/or molecules designed toincrease KLK6 polypeptide activity can be used to promote cardiac,skeletal, or vascular muscle cell survival. For example, KLK6polypeptides can be used to promote bone cell survival, therebypreventing osteoporosis and/or promoting bone and cartilage repair andregeneration. In some cases, KLK6 polypeptides, molecules designed toincrease KLK6 polypeptide expression, and/or molecules designed toincrease KLK6 polypeptide activity can be used to reduce cell death thatoccurs with aging.

In some cases, conditions or exposures known to induce apoptosis (e.g.,withdrawal of growth factors, activation of cell surface deathreceptors, exposure to heat shock, hypoxia, UV radiation, DNA damage,viral infection, dexamethasone, cytotoxic agents, or chemotherapeuticagents) can be treated by administering KLK6 polypeptides, moleculesdesigned to increase KLK6 polypeptide expression, and/or moleculesdesigned to increase KLK6 polypeptide activity. For example, acomposition containing KLK6 polypeptides can be used to treat severesunburns, thereby reducing the level of apoptosis that occurs. In somecases, cancer therapeutics such as chemotherapeutic agents known toinduce apoptosis of cells (e.g., mucosal membrane cells, lymphocytes,leukocytes, and hair follicles) can be administered with KLK6polypeptides, molecules designed to increase KLK6 polypeptide expressionlevels, and/or molecules designed to increase KLK6 polypeptide activityto reduce the level of general apoptosis induced by the cancertherapeutic. Examples of such cancer therapeutics include, withoutlimitation, alkylating agents such as Cisplatin, vinca alkaloids such asVincristine, Taxanes such as Taxonl, topoisomerase inhibitors such ascamptothecin and topotecan, anti-neoplastics such as doxorubicin, andanti-metabolites. In such cases, the composition including a KLK6polypeptide, molecule designed to increase KLK6 polypeptide expression,and/or molecule designed to increase KLK6 polypeptide activity can beadministered prior to, with, or after administration of the cancertherapeutic.

In some cases, KLK6 polypeptides, molecules designed to increase KLK6polypeptide expression, and/or molecules designed to increase KLK6polypeptide activity can be used to inhibit the pro-apoptotic proteinBim and to enhance levels of the anti-apoptotic protein Bcl-XL.

In some cases, cells within a mammal can be exposed a compositioncontaining KLK6 polypeptides to promote resistance to apoptosis. In somecases, the level of KLK6 polypeptide expression or activity can beincreased by administering a KLK6 polypeptide agonist or a nucleic acidencoding a KLK6 polypeptide. Such a nucleic acid can encode afull-length KLK6 polypeptide such as a human KLK6 polypeptide having theamino acid sequence set forth in FIG. 22, or a biologically activefragment of a KLK6 polypeptide having amino acid residues 22 to 244 ofthe sequence set forth in FIG. 22. See, also, GenBank® Accession No.NM_(—)002774 (GI No. 61744422); Bernett et al., J. Biol. Chem.,277:24562-24570 (2002) and Blaber et al., Biochemistry, 41:1165-1173(2002)). A nucleic acid encoding a KLK6 polypeptide or fragment thereofcan be administered to a mammal using any appropriate method. Forexample, a nucleic acid can be administered to a mammal using a vectorsuch as a viral vector.

Vectors for administering nucleic acids (e.g., a nucleic acid encoding aKLK6 polypeptide or fragment thereof) to a mammal are known in the artand can be prepared using standard materials (e.g., packaging celllines, helper viruses, and vector constructs). See, for example, GeneTherapy Protocols (Methods in Molecular Medicine), edited by Jeffrey R.Morgan, Humana Press, Totowa, N.J. (2002) and Viral Vectors for GeneTherapy: Methods and Protocols, edited by Curtis A. Machida, HumanaPress, Totowa, N.J. (2003). Virus-based nucleic acid delivery vectorsare typically derived from animal viruses, such as adenoviruses,adeno-associated viruses, retroviruses, lentiviruses, vaccinia viruses,herpes viruses, and papilloma viruses. Lentiviruses are a genus ofretroviruses that can be used to infect cells (e.g., cancer cells).Adenoviruses contain a linear double-stranded DNA genome that can beengineered to inactivate the ability of the virus to replicate in thenormal lytic life cycle. Adenoviruses and adeno-associated viruses canbe used to infect cancer cells.

Vectors for nucleic acid delivery can be genetically modified such thatthe pathogenicity of the virus is altered or removed. The genome of avirus can be modified to increase infectivity and/or to accommodatepackaging of a nucleic acid, such as a nucleic acid encoding a KLK6polypeptide or fragment thereof. A viral vector can bereplication-competent or replication-defective, and can contain fewerviral genes than a corresponding wild-type virus or no viral genes atall.

In addition to nucleic acid encoding a KLK6 polypeptide or fragmentthereof, a viral vector can contain regulatory elements operably linkedto a nucleic acid encoding a KLK6 polypeptide or fragment thereof. Suchregulatory elements can include promoter sequences, enhancer sequences,response elements, signal peptides, internal ribosome entry sequences,polyadenylation signals, terminators, or inducible elements thatmodulate expression (e.g., transcription or translation) of a nucleicacid. The choice of element(s) that may be included in a viral vectordepends on several factors, including, without limitation, inducibility,targeting, and the level of expression desired. For example, a promotercan be included in a viral vector to facilitate transcription of anucleic acid encoding a KLK6 polypeptide or fragment thereof. A promotercan be constitutive or inducible (e.g., in the presence oftetracycline), and can affect the expression of a nucleic acid encodinga KLK6 polypeptide or fragment thereof in a general or tissue-specificmanner. Tissue-specific promoters include, without limitation, enolasepromoter, prion protein (PrP) promoter, and tyrosine hydroxylasepromoter.

As used herein, “operably linked” refers to positioning of a regulatoryelement in a vector relative to a nucleic acid in such a way as topermit or facilitate expression of the encoded polypeptide. For example,a viral vector can contain a neuronal-specific enolase promoter and anucleic acid encoding a KLK6 polypeptide or fragment thereof. In thiscase, the enolase promoter is operably linked to a nucleic acid encodinga KLK6 polypeptide or fragment thereof such that it drives transcriptionin neuronal cells.

A nucleic acid encoding a KLK6 polypeptide or fragment thereof also canbe administered to cells using non-viral vectors. Methods of usingnon-viral vectors for nucleic acid delivery are known to those ofordinary skill in the art. See, for example, Gene Therapy Protocols(Methods in Molecular Medicine), edited by Jeffrey R. Morgan, HumanaPress, Totowa, N.J. (2002). For example, a nucleic acid encoding a KLK6polypeptide or fragment thereof can be administered to a mammal bydirect injection of nucleic acid molecules (e.g., plasmids) comprisingnucleic acid encoding a KLK6 polypeptide or fragment thereof, or byadministering nucleic acid molecules complexed with lipids, polymers, ornanospheres.

A nucleic acid encoding a KLK6 polypeptide or fragment thereof can beproduced by standard techniques, including, without limitation, commonmolecular cloning, polymerase chain reaction (PCR), chemical nucleicacid synthesis techniques, and combinations of such techniques. Forexample PCR or RT-PCR can be used with oligonucleotide primers designedto amplify nucleic acid (e.g., genomic DNA or RNA) encoding a KLK6polypeptide or fragment thereof. In some cases, the methods describedelsewhere can be used to make or use nucleic acid encoding a KLK6polypeptide or biologically active fragment thereof (see, e.g., Bernettet al., J. Biol. Chem., 277:24562-24570 (2002) and Blaber et al.,Biochemistry, 41:1165-1173 (2002)).

This document also provides methods and materials for reducing theability of KLK6 polypeptides to promote resistance to apoptosis. Forexample, an inhibitor of KLK6 polypeptide activity can be used to reduceKLK6 polypeptide-induced resistance to apoptosis. As described herein,KLK6 polypeptide inhibitors and molecules designed to reduce KLK6polypeptide expression can be used to treat conditions and diseases thatexhibit undesirable resistance to apoptosis. Examples of KLK6polypeptide inhibitors include, without limitation, KLK6 antisensemolecules, KLK6 small hairpin RNA target molecules, KLK6-specific miRNAmolecules, function blocking anti-KLK6 antibodies (e.g., mK6-2 and mK6-3antibodies (Blaber et al., FASEB J., 19:920-922 (2004)), pharmacologicKLK6-specific inhibitors, and combinations thereof. Examples of KLK6small hairpin RNA target molecules include, without limitation,GAGCAGAATAAG-TTGGTGCAT (SEQ ID NO:3), CCTCTACACCTCGGGCCACTT (SEQ IDNO:4), AGCCAAACTCTCTGAACTCAT (SEQ ID NO:5), and GATGAGAAGTACG-GGAAGGAT(SEQ ID NO:6). See, also, Henkhaus et al., Biological Chemistry, 389:757-764 (2008)). Examples of KLK6-specific miRNA molecules include,without limitation, hsa-let-7f-1 and hsa-let-7f-2 (Chow et al.,Biological Chemistry, 389:731-738 (2008)). In some cases, blockade ofthe receptor complement that KLK6 activates, which is known to includeprotease activated receptors 1 (PAR1) and PAR2 and bradykinin receptor 2(B2) can be used to reduce apoptosis resistance.

Antibodies having specific binding affinity for a KLK6 polypeptide canbe used to inhibit KLK6 polypeptide activity (e.g., decrease activity).As used herein, the terms “antibody” or “antibodies” include intactmolecules as well as fragments thereof that are capable of binding to anepitopic determinant of a KLK6 polypeptide (e.g., human KLK6polypeptide). The term “epitope” refers to an antigenic determinant onan antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains, and typically havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. Epitopes generally have at least fivecontiguous amino acids (a continuous epitope), or alternatively can be aset of noncontiguous amino acids that define a particular structure(e.g., a conformational epitope). The terms “antibody” and “antibodies”include polyclonal antibodies, monoclonal antibodies, humanized orchimeric antibodies, single chain Fv antibody fragments, Fab fragments,and F(ab)₂ fragments. Polyclonal antibodies are heterogenous populationsof antibody molecules that are contained in the sera of the immunizedanimals. Monoclonal antibodies are homogeneous populations of antibodiesto a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for a KLK6polypeptide can be generated by known techniques. For example, F(ab′)₂fragments can be produced by pepsin digestion of the antibody molecule;Fab fragments can be generated by reducing the disulfide bridges ofF(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed. See, for example, Huse et al., Science, 246:1275 (1989).Once produced, antibodies or fragments thereof are tested forrecognition of a KLK6 polypeptide by standard immunoassay methodsincluding ELISA techniques, radioimmunoassays, and Western blotting.See, Short Protocols in Molecular Biology, Chapter 11, Green PublishingAssociates and John Wiley & Sons, Edited by Ausubel, F. M et al., 1992.

Antibodies having specific binding affinity for a KLK6 polypeptide canbe produced through standard methods. In general, a KLK6 polypeptide canbe recombinantly produced, or can be purified from a biological sample,and used to immunize animals. To produce a recombinant KLK6 polypeptide,a nucleic acid sequence encoding a KLK6 polypeptide can be ligated intoan expression vector and used to transform a bacterial or eukaryotichost cell. Nucleic acid constructs typically include a regulatorysequence operably linked to a KLK6 nucleic acid sequence. Regulatorysequences do not typically encode a gene product, but instead affect theexpression of the nucleic acid sequence. In bacterial systems, a strainof Escherichia coli such as BL-21 can be used. Suitable E. coli vectorsinclude the pGEX series of vectors that produce fusion proteins withglutathione S-transferase (GST). Transformed E. coli are typically grownexponentially, then stimulated with isopropylthiogalactopyranoside(IPTG) prior to harvesting. In general, such fusion proteins are solubleand can be purified easily from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

Mammalian cell lines that stably express a KLK6 polypeptide can beproduced by using expression vectors with the appropriate controlelements and a selectable marker. For example, the eukaryotic expressionvector pCDNA.3.1+ (Invitrogen, San Diego, Calif.) is suitable forexpression of a KLK6 polypeptide in, for example, COS cells, Chinesehamster ovary (CHO), or HEK293 cells. Following introduction of theexpression vector by electroporation, DEAE dextran, or other suitablemethod, stable cell lines are selected. Alternatively, a KLK6polypeptide can be transcribed and translated in vitro using wheat germextract or rabbit reticulocyte lysase.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express a KLK6 polypeptide. A nucleic acid encoding aKLK6 polypeptide can be introduced into a SV40, retroviral or vacciniabased viral vector and used to infect host cells.

Various host animals can be immunized by injection of a KLK6polypeptide. Host animals include, without limitation, rabbits,chickens, mice, guinea pigs, and rats. Various adjuvants that can beused to increase the immunological response depend on the host speciesand include Freund's adjuvant (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface-active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies canbe prepared using a KLK6 polypeptide and standard hybridoma technology.In particular, monoclonal antibodies can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture such as described by Kohler et al., Nature,256:495 (1975), the human B-cell hybridoma technique (Kosbor et al.,Immunology Today, 4:72 (1983); Cole et al., Proc. Natl. Acad. Sci. USA,80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss, Inc., pp.77-96 (1983)). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Thehybridoma producing the monoclonal antibodies of the invention can becultivated in vitro and in vivo.

In some embodiments, anti-KLK6 antibodies can inhibit the enzymaticactivity of a KLK6 polypeptide. In vitro assays can be used to monitorKLK6 polypeptide activity after incubation in the presence of anantibody. Typically, a KLK6 polypeptide can be incubated with anantibody (e.g., polyclonal or monoclonal), then the ability of the KLK6polypeptide to cleave a substrate such as myelin basic protein or anarginine-specific fluorogenic substrate can be assessed at 37° C. in asuitable buffer (e.g., Tris buffer). Depending on the substrate,cleavage can be monitored using sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) or a spectrophotometer.

Antisense oligonucleotides can be used to reduce expression of a KLK6polypeptide. A KLK6 antisense oligonucleotide can be at least 8nucleotides in length. For example, a KLK6 antisense oligonucleotide canbe about 8, 9, 10-20 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleotides in length), 15 to 20, 18-25, or 20-50 nucleotides in length.In other embodiments, KLK6 antisense oligonucleotides can be used thatare greater than 50 nucleotides in length, including the full-lengthsequence of a KLK6 mRNA. As used herein, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or analogs thereof Nucleic acid analogs canbe modified at the base moiety, sugar moiety, or phosphate backbone toimprove, for example, stability, hybridization, or solubility of anucleic acid. Modifications at the base moiety include substitution ofdeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Other examples ofnucleobases that can be substituted for a natural base include5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other usefulnucleobases include those disclosed, for example, in U.S. Pat. No.3,687,808.

Modifications of the sugar moiety can include modification of the 2′hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars.The deoxyribose phosphate backbone can be modified to produce morpholinonucleic acids, in which each base moiety is linked to a six-membered,morpholino ring, or peptide nucleic acids, in which the deoxyphosphatebackbone is replaced by a pseudopeptide backbone (e.g., anaminoethylglycine backbone) and the four bases are retained. See, forexample, Summerton and Weller, Antisense Nucleic Acid Drug Dev.,7:187-195 (1997); and Hyrup et al., Bioorgan. Med. Chem., 4:5-23 (1996).In addition, the deoxyphosphate backbone can be replaced with, forexample, a phosphorothioate or phosphorodithioate backbone, aphosphoroamidite, or an alkyl phosphotriester backbone. See, forexample, U.S. Pat. Nos. 4,469,863, 5,235,033, 5,750,666, and 5,596,086for methods of preparing oligonucleotides with modified backbones.

In some cases, KLK6 antisense oligonucleotides can be modified bychemical linkage to one or more moieties or conjugates that enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties (e.g., a cholesterol moiety); cholic acid; a thioether moiety(e.g., hexyl-S-tritylthiol); a thiocholesterol moiety; an aliphaticchain (e.g., dodecandiol or undecyl residues); a phospholipid moiety(e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate); a polyamine or apolyethylene glycol chain; adamantane acetic acid; a palmityl moiety; oran octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Thepreparation of such oligonucleotide conjugates is disclosed in, forexample, U.S. Pat. Nos. 5,218,105 and 5,214,136.

Methods for synthesizing antisense oligonucleotides are known, includingsolid phase synthesis techniques. Equipment for such synthesis iscommercially available from several vendors including, for example, LifeTechnologies (AKA, Applied Biosystems; Foster City, Calif.).Alternatively, expression vectors that contain a regulatory element thatdirects production of an antisense transcript can be used to produceantisense molecules.

KLK6 antisense oligonucleotides can bind to a nucleic acid encoding aKLK6 polypeptide, including DNA encoding KLK6 RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA, under physiological conditions (i.e., physiological pH and ionicstrength). The nucleic acid sequence encoding a human KLK6 polypeptidecan be found in GenBank® under Accession No. AF013988 (GI No. 2318114),AF149289 (GI No. 5791635), D78203 (GI No. 1805492), or NM_(—)002774 (GINo. 61744422). The nucleic acid sequence encoding a rat KLK6 polypeptidecan be found in GenBank® under Accession No. AF016269 (GI No. 2853365).The nucleic acid sequence encoding a mouse KLK6 polypeptide can be foundin GenBank® under Accession No. NM_(—)019175.1 (GI No. 9506996). Forexample, an antisense oligonucleotide can hybridize under physiologicalconditions to the nucleotide sequence set forth in GenBank Accession No.AF013988, AF149289, 78203, NM_(—)002774, AF016269, or NM_(—)019175.1.

is understood in the art that the sequence of an antisenseoligonucleotide need not be 100% complementary to that of its targetnucleic acid to be hybridizable under physiological conditions.Antisense oligonucleotides hybridize under physiological conditions whenbinding of the oligonucleotide to the KLK6 nucleic acid interferes withthe normal function of the KLK6 nucleic acid, and non-specific bindingto non-target sequences is minimal.

Target sites for KLK6 antisense oligonucleotides include the regionsencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. In addition, the ORF has been targetedeffectively in antisense technology, as have the 5′ and 3′ untranslatedregions. Furthermore, antisense oligonucleotides have been successfullydirected at intron regions and intron-exon junction regions. Furthercriteria can be applied to the design of antisense oligonucleotides.Such criteria are well known in the art, and are widely used, forexample, in the design of oligonucleotide primers. These criteriainclude the lack of predicted secondary structure of a potentialantisense oligonucleotide, an appropriate G and C nucleotide content(e.g., approximately 50%), and the absence of sequence motifs such assingle nucleotide repeats (e.g., GGGG runs). The effectiveness ofantisense oligonucleotides at modulating expression of a KLK6 nucleicacid can be evaluated by measuring levels of the KLK6 mRNA orpolypeptide (e.g., by Northern blotting, RT-PCR, Western blotting,ELISA, or immunohistochemical staining)

mples of conditions that can be treated by promoting apoptosis viainhibition of KLK6 polypeptide expression or activity include, withoutlimitation, inflammatory conditions (e.g., rheumatoid arthritis, Crohn'sdisease, multiple sclerosis, systemic lupus erythematosus, andpsoriasis), lymphoproliferative diseases (e.g., follicular lymphoma,chronic lymphocytic leukemia, acute lymphoblastic leukemia, hair cellleukemia, lymphomas, multiple myeloma, Waldenstrom's macroglobulinemia,Wiskott-Aldrich syndrome, post-transplant lymphoproliferative disorder,Autoimmune lymphoproliferative syndrome, and lymphoid interstitialpneumonia), cancer (e.g., solid tumor cancers and blood cancers),infectious diseases (e.g., viral, bacterial, fungal, parasitic,protozoal, and prion infections), and cardiovascular diseases (e.g.,coronary heart disease, cardiomyopathy, ischemic heart disease, heartfailure, hypertensive heart disease, inflammatory heart disease, andvalvular heart disease). Since KLK6 polypeptides exhibit pro-survivaleffects on human glioma cells, inhibition of KLK6 polypeptide can beused to reduce tumor cell survival, thereby promoting tumor regressionand making tumor cells more susceptible to current therapies, includingradiation and chemotherapy. In some cases, KLK6 polypeptide inhibitorscan be used to increase apoptosis in solid tumors such as gliomas, lungcancer, bladder cancer, breast cancer, colon cancer, head and neckcancer, pancreatic cancer, cervical cancer, prostate cancer, and skincancer, or blood cancers such as leukemias and lymphomas. In some cases,reducing KLK6 polypeptide levels or activity can be used to improveapoptosis for application in tissue remodeling and/or tissueregeneration such as organ/bone repair, scar reduction, or fat cellremoval. In some cases, a lymphoproliferative disease, cancer,infection, or cardiovascular disease treated as described herein with aKLK6 polypeptide inhibitor can be a non-inflammatory condition.

Any appropriate method can be used to administer a KLK6 polypeptideinhibitor to reduce resistance to apoptosis. For example, an effectiveamount of a composition containing KLK6 polypeptide inhibitor can beadministered to a mammal by any route, including, without limitation,oral or parenteral routes of administration such as intravenous,intramuscular, intraperitoneal, subcutaneous, intrathecal,intraarterial, nasal, transdermal (e.g., as a patch), or pulmonaryabsorption. An effective amount of a KLK6 polypeptide inhibitor can bean amount that reduces resistance to apoptosis without inducingsignificant toxicity to the host. Effective amounts of KLK6 polypeptideinhibitors can be determined by a physician, taking into account variousfactors that can modify the action of drugs such as overall healthstatus, body weight, sex, diet, time and route of administration, othermedications, and any other relevant clinical factors.

A KLK6 polypeptide inhibitor can be formulated as, for example, asolution, suspension, or emulsion with pharmaceutically acceptablecarriers or excipients suitable for the particular route ofadministration, including sterile aqueous or non-aqueous carriers.Aqueous carriers include, without limitation, water, alcohol, saline,and buffered solutions. Examples of non-aqueous carriers include,without limitation, propylene glycol, polyethylene glycol, vegetableoils, and injectable organic esters. Preservatives, flavorings, sugars,and other additives such as antimicrobials, antioxidants, chelatingagents, inert gases, and the like also may be present.

For oral administration, tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g. magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). Tablets can be coated by methods known in the art.Preparations for oral administration can also be formulated to givecontrolled release of the compound.

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulised aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

In some embodiments, anti-cancer agents can be administered incombination with a KLK6 polypeptide inhibitor. For example, ananti-cancer agent such as temazolamide, avastin, taxol, or radiation canbe administered to a mammal together with a KLK6 polypeptide inhibitor.

This document also provides methods and materials related to identifyingagonists or antagonists of KLK6 polypeptide activity (e.g., pro-survivalactivity). For example, candidate molecules can be screened to identifyKLK6 polypeptide inhibitors that can block the ability of KLK6polypeptides to rescue cells from apoptosis induced by staurosporine,camptothecin, and/or dexamethasone as described herein. See, e.g., FIGS.1-21.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Kallikrein 6 Polypeptides Promote Resistance ofLymphocytes to Apoptosis Model Systems

The effect of KLK6 polypeptides on immune cell survival was examinedusing whole splenocyte populations derived from C57BL6/J or PARIdeficient mice or using the Jurkat T-leukemic cell line (clone E6-1,TIB-152 American Type Culture Collection). Eight to 12 week old C57 micewere obtained from Jackson Laboratories. Mice deficient in PARI ^(−/−)(B6.129S4-F2r^(tm1Ajc)/J) were also obtained from Jackson andbackcrossed to C57BL6/J for 10 generations such that C57 mice served aswild type controls in each experiment.

Cell Culture

Spleens were homogenized in RPMI-1640, red blood cells lysed withammonium chloride buffer, and splenocytes cultured in tissue culturetreated 96 well plates at a density of 7.5×10⁵ cells/mL. All experimentswere performed in serum free X-Vivo media (Lonza, Mapleton, Ill.)containing 2 mM Glutamax, 1 mM sodium pyruvate, 50 U/mLpenicillin-streptomycin, 10 mM HEPES, 50 μM 2-β-mercaptoethanol, and 10μM non-essential amino acids (Invitrogen). Jurkat T cells weremaintained in log-phase growth in the same defined media. The cells weremaintained at 37° C. in 95% air and 5% CO2. The culture conditions wereexamined in triplicate within a given experiment, and all experimentsrepeated at least twice.

Reagents

Recombinant KLK1 polypeptides and KLK6 polypeptides were expressed usinga baculovirus/insect system, purified and activated as describedelsewhere (Bernett et al., J. Biol. Chem., 277:24562-24570 (2002); andLaxmikanthan et al., Proteins, 58:802-814 (2005)). Thrombin derived frombovine serum was obtained from Sigma (St. Louis, Mo.) and used at 50 nM.PAR1, PAR2 and PAR4-activating polypeptides (-APs) were obtained fromPeptides International (Louisville, Ky.). Recombinant KLK polypeptideswere used at concentrations ranging from 1 to 10 μg/mL (40 to 400 nM),and PAR-APs were used at 100 μM, concentrations shown elsewhere toinduce intracellular signaling (Vandell et al., J. Neurochem.,107:855-870 (2008)). The concentrations of KLK6 polypeptides examinedencompass the physiologic level seen in normal cerebrospinal fluid(0.5-2 μg/mL) (Diamandis et al., Clin. Biochem., 33:579-583 (2000);Zarghooni et al., Clinical Biochemistry, 35:225-231 (2002); and Borgonoet al., Mol. Cancer Res., 2:257-280 (2004)) and 5-fold excess whichmodels elevated levels seen at sites of CNS inflammation in MS and itsanimal models (Scarisbrick et al., Brain, 125:1283-1296 (2002);Christophi et al., J. Neurochem., 91:1439-1449 (2004); Scarisbrick etal., J. Neuroimmunology, 178:167-176 (2006); Scarisbrick et al., Biol.Chem., 389:739-745 (2008)).

To delineate the scope of action of KLK6 polypeptides on immune cellsurvival, its effects on cell survival were examined in severaldifferent cell death paradigms encompassing both the intrinsic andextrinsic cell death pathways. First, spontaneous cell death, which isknown to occur after harvest and plating of splenocytes in vitro andwhich is referred to as resting cell death, was examined. In addition,cell death was induced using the topoisomerase inhibitor camptothecin(1.0 μM), the mitogen concanavalin A (ConA, 5 μg/mL), or a combinationof ConA and camptothecin. Fas ligand cell death was induced using FasLigand/TNFSF6 (2 μg/mL) cross linked with anti-polyHistidine (10 μg/mL).Cell death due to stimulation by the glucocorticoid Dexamethasone (0.1μM) and by the protein kinase inhibitor staurosporine (1 μM) were alsoexamined. All agents to induce cell death were obtained from Sigma andwere either applied to cultures at the time of cell platingsimultaneously with KLK6 polypeptides, or after cultures had first beenpre-incubated with KLK6 polypeptides for 2 hours.

Flow Cytometry

Following experimental incubation periods, which ranged from 4 to 72hours, cells were harvested and stained with combinations of antibodiesrecognizing to CD45, CD3, B220, or the early apoptotic marker Annexin-V,conjugated to FITC, PE, or APC (e-Bioscience, San Diego, Calif.). Ineach case, either propidium iodide (PI) or 7-AAD were used to label deadcells (Sigma). Cells were analyzed by flow cytometry using a FACSCaliburflow cytometer (BD Biosciences, Mountain View, Calif.). Live immunecells were defined as those which were positive for the common leukocyteantigen CD45, but negative for PI. Early and late apoptotic/dead cellswere distinguished using a combination of AnnexinV-PE and 7-AAD orAnnexnV-FITC and PI (BD Biosciences). FlowJo software (Ashland, Oreg.)was used to quantify different cell populations in flow cytometryexperiments. Potential effects on cell proliferation were examined bylabeling cells with carboxyfluorescein succinimidyl ester (CFSE,Invitrogen, Carlsbad, Calif.) prior to plating followed by analysisusing the FlowJo proliferation platform at the completion of eachexperiment.

To compare multiple cell culture conditions within a given experiment,the mean and standard error across experimental groups were analyzed byOne-Way Analysis of Variance followed by Student-Newman-Keuls (SNK) posthoc test except when data were not normally distributed when ANOVA onRanks and Mann-Whitney-U analyses were performed. Statisticaldifferences between two groups were compared using the Student's t-testor the Rank Sum test when data were non-linear. P<0.05 was consideredstatistically significant.

Western Blot Analysis

Protein lysates were separated on SDS-polyacrylaminde gels prior totransfer to nitrocellulose membranes. Antibodies to detect Bcl-XL(B-cell lymphoma-extra large) and Bim (Bcl-2-interacting mediator ofcell death) polypeptides were obtained from Cell Signaling Technology(Danvers, Mass.). The PARP antibody was obtained from Dr. S. Kauffman(Eischen et al., Blood, 90:935-943 (1997)). Equal loading was verifiedby re-probing blots for β-Actin (Novus, Littleton, Colo.). In all cases,polypeptides of interest were detected using chemiluminescence (Pierce,Rockford, Ill.). All Western blots were repeated at least three timesusing separate cell culture preparations with similar results.

Differential Effects of KLKI and KLK6 Polypeptides on Immune cellProliferation and Survival

As a first approach to define the possible immunological actions of KLK6polypeptides, its effects on proliferation and survival of murinesplenocytes were examined and compared to another kallikrein familymember, KLK1. While KLK6 polypeptides did not significantly altersplenocyte proliferation after 24 or 72 hours, KLK6 polypeptidespromoted a substantial and dose dependent reduction in the number ofcells positive for markers of cell death across the same time points(FIG. 1, see also FIGS. 4, 5, 6, 7, 9, and 11). KLK6 polypeptidessignificantly reduced cell death under resting conditions when pulsed ateither 1 or 10 μg/mL at the time of plating. Ten μg/mL of KLK6polypeptides generated significantly better rescue than 1 μg/mL (FIG.1B) and therefore all subsequent experiments were performed using thehigher concentration. Also, KLK6 polypeptides promoted cell survival atboth 24- and 72-hour time points such that most subsequent experimentswere carried out to 24 hours. Parallel examination of a secondkallikrein, KLK1, produced in an identical fashion to KLK6 (Laxmikanthanet al., Proteins, 58:802-814 (2005)), did not significantly impact cellsurvival. KLK1 polypeptides however promoted a significant increase inthe percent of divided cells when pulsed with 10 μg/mL and examined at24 hours post-stimulation. Significant differences in proliferation werenot seen at lower concentrations of KLK1, or when cells were examined 72hours post-exposure. KLK6 polypeptides did not produce similar robusteffects on cell proliferation at any of the concentrations or timepoints examined indicating proliferative effects are unlikely to accountfor its ability to increase the number of viable cells.

KLK6 Polypeptides Protect T Cells and B Cells Across Multiple Cell DeathParadigms

To determine how robust the survival promoting effects of KLK6polypeptides are, the ability of KLK6 polypeptides to prevent cell deathacross a range of paradigms was examined. KLK6 polypeptides not onlyprevented the death of splenocytes that occurred after explant andculture (FIGS. 1 and 4-7), but also that seen in response to 0.1 μMdexamethasone (FIGS. 4-7), in the presence of graded concentrations ofFas ligand (FIG. 4), and after exposure to 1 μM staurosporine (FIG. 6).KLK6 polypeptides also blocked the death of Jurkat T cells under restingconditions and when exposed to 1 μM camptothecin, 1 μM camptothecin plus5 μg/mL ConA, or Fas ligand receptor cross linking (FIGS. 4 and 11).Co-labeling splenocytes with markers for T or B cell subsets in additionto markers for cell death demonstrated that KLK6-rescue effects occurredacross both populations (FIGS. 4 and 7). The ability of KLK6polypeptides to rescue splenocytes or Jurkat cells across cell deathparadigms was consistently observed when KLK6 polypeptides were appliedsimultaneously with death inducing agents. A two hour pre-incubationwith KLK6 polypeptides prior to dexamethasone application was shown toresult in a small but statistically significant enhancement of survival(FIG. 4, P<0.05).

Additional results include using ATP and cisplatin with murine T and Blymphocytes to demonstrate that recombinant KLK6 blocks apoptosis. KLK6protected immune cells from cellular injury and death even in the faceof a strong apoptosis inducing agent (ATP) or a strong chemotherapeuticagent (Cisplatin) (FIGS. 2 and 3).

As Little as a 5-Minute Exposure to KLK6 Polypeptides was Sufficient toPromote Robust Survival

To determine whether prolonged or continual exposure to KLK6polypeptides was necessary to promote significant rescue effects, thepotential pro-survival effects of abbreviated periods of KLK6polypeptide-stimulation were examined (FIG. 5). Under both restingconditions and in the presence of dexamethasone, as little as a 5-minutepulse with KLK6 polypeptides was sufficient to reduce cell deathsignificantly when cells were subsequently cultured for an additional 24hours in the absence of KLK6 polypeptides. In the presence ofdexamethasone for 24 hours, significant rescue effects were also seenwith 5-, 30-, or 60-minute pulses of KLK6 polypeptides, and themagnitude of rescue did not differ significantly from that seen withcontinual KLK6 polypeptide exposure over the full period ofdexamethasone treatment. Experimental endpoints under resting conditionswere also extended to 48 hours, and in this case, while continual KLK6polypeptide stimulation promoted robust survival, KLK6 polypeptidepulses up to 60 minutes in length were insufficient to inducesignificant rescue.

KLK6 Polypeptides Halt the Apoptotic Cascade

The ability of KLK6 polypeptides to specifically affect apoptosis insplenocytes and Jurkat cells was assessed by comparing KLK6polypeptide-induced changes in the relative number of live cells(AnnexinV−, PI−), early apoptotic cells (AnnexinV+, PI−), and dead cells(AnnexinV+, PI+) across several cell death paradigms (FIGS. 6 and 11).In some cases, 7-AAD was used in place of PI.

In the case of Jurkat T cells under resting conditions, KLK6polypeptides promoted a reduction in the number of dead cells and acorresponding increase in the number of live cells, but did notsignificantly alter the number of early apoptotic cells when examined at4, 24, or 48 hours post plating (FIGS. 11). When Jurkat cells werechallenged with the topoisomerase inhibitor camptothecin, with ConA, ora combination of these agents, KLK6 polypeptides reduced the number ofdead cells at the 4-, 24-, and 48-hour time points examined, mirroringobservations under resting conditions. A significant increase in thenumber of live cells was also seen in each circumstance except after48-hour exposure to the Camptothecin or the ConA+Camptothecincombination. Jurkat cells cultured in the presence of KLK6 in additionto ConA, or ConA plus camptothecin, showed an accumulation of cells atearly apoptotic stages at the 24-hour time point examined. By 48 hours,the significant increase in early apoptotic cells induced by thepresence of KLK6 polypeptides was seen under all three death-inducingconditions, including camptothecin alone.

In addition, over expression of KLK6 polypeptides in the Jurkat leukemiaT cell line promoted the resistance of these cells to apoptosis underresting conditions or in the presence of staurosporine (FIG. 12).

Examination of live, early apoptotic, and dead cells in murinesplenocyte cultures generated results parallel to those observed inJurkat cells. That is, KLK6 polypeptides reduced overall cell death byincreasing the number of cells in the live and/or early apoptoticpopulations depending on the conditions examined (FIGS. 6A). Underresting conditions over 24 hours, the presence of KLK6 polypeptidessignificantly reduced the number of dead cells with a correspondingincrease in the live population, while the number of early apoptoticcells was largely unchanged. In the presence of dexamethasone (0.1 μM)or staurosporine (1 μM), co-exposure to KLK6 polypeptides promoted theexpected significant decrease in the number of dead cells and a smallbut significant increase in the live cell population. In addition, underthese death-inducing conditions, KLK6 polypeptides also promoted asubstantial and significant increase in the number of early apoptoticcells.

The ability of KLK6 polypeptides to block apoptosis was further assessedby examining the effects of KLK6 polypeptides on cleavage of poly-ADPribose polymerase (PARP), one of the final caspase substrates cleaved inthe apoptotic cascade (FIG. 6B). Significant levels of cleaved PARP weredetected in splenocytes grown under resting conditions and when exposedto camptothecin for 24 hours. In each case, co-exposure to KLK6polypeptides significantly reduced the amount of cleaved PARP detected.

KLK6 Polypeptide-Mediated Lymphocyte Survival Depends in Part onActivation of PAR1

To investigate the potential involvement of PAR1 polypeptides in KLK6polypeptide-mediated lymphocyte rescue, the impact of PAR1 polypeptidedeficiency was determined using splenocytes isolated from PAR1 knockoutmice. Additionally, the effects of KLK6 polypeptides were compared tocommercially available short synthetic PAR-derived peptide sequencesthat selectively activate PAR1, 2, or 4 (PAR-APs) (FIG. 7). In the caseof CD3+ T cells, the absence of PAR1 severely impaired the ability ofKLK6 polypeptides to rescue cells under resting conditions but anyeffect in the presence of dexamethasone was not statisticallysignificant (FIG. 7A). PAR1 deficiency also blocked the ability of KLK6polypeptides to rescue B220+ B cells from cell death under restingconditions and reduced KLK6 polypeptide-mediated rescue in the presenceof dexamethasone (FIG. 7B). While these results indicate that KLK6polypeptide-mediated lymphocyte rescue is dependent at least in part onthe presence of PAR1, activation of PAR1 or PAR2 alone using PAR-APs, orin combination with each other (not shown), or in combination withPAR4-AP, were not sufficient to recapitulate the rescue effects seenwith KLK6 polypeptides (FIGS. 7C and 7D). Indeed, in the presence ofdexamethasone, PAR1-AP enhanced the magnitude of cell death observed(FIG. 7C).

In murine lymphocytes, deletion of protease activated receptor 2 (PAR2)(FIG. 8), like PAR1 (FIG. 7), diminished the ability of KLK6 to promotecell survival in resting cells and those treated with the corticosteroiddexamethasone. Thus, targeting PAR1 and/or PAR2 is another potentialmechanism to target (i.e., increase or decrease) the activity of KLK6.

KLK6 Polypeptides Differentially Regulate Bcl-2 Family Member Signalingin a PAR1-Dependent Fashion

To further delineate the mechanism by which KLK6 polypeptides preventcell death, effects on pro- and anti-apoptotic signaling pathways wereexamined. When splenocytes were exposed to either camptothecin or ConAin the presence of KLK6 polypeptides, significant elevations in thepro-survival protein Bcl-XL were observed (FIG. 9). As a corollary,under resting conditions, or in the presence of camptothecin, or ConA,KLK6 polypeptides promoted significant reductions in the pro-apoptoticprotein Bim. The ability of KLK6 polypeptides to alter these signalingcascades was largely blocked in mice deficient in PAR1.

Example 2 KLK6 Polypeptides Promote the Survival of T Cells, Monocytes,and B Cells

Murine T cells (CD3+), monocytes (CD11b+), and B cells (B220+) eachundergo cell death when harvested from the mouse spleen and grown inculture for 24 hours. When exposed to 10 μg/mL of recombinant KLK6polypeptides at the time of plating, a significant reduction in theamount of cell death was observed (FIG. 10; P<0.05, ANOVA, SNK post hoctest). Cell death was measured by flow cytometric analysis of PIlabeling.

Example 3 KLK6 Polypeptides Promote the Survival of Oligodendrocytes

KLK6 polypeptides promoted the survival of murine oligodendrocytes in adose dependent fashion. The protein tyrosine kinase inhibitor,staurosporine (0.5 μM), was shown to promote death of the Oli Neuoligodendrocyte cell line after an 18 hour exposure period. Cell deathwas detected by PI labeling and quantitatively analyzed by flowcytometry. When cells were pre-exposed to 5 or 10 μg/mL of recombinantKLK6 polypeptides prior to stuarosporine exposure, a significantreduction in the amount of cell death was observed (FIG. 13; P<0.001,One Way ANOVA, SNK post hoc test).

Example 4 KLK6 Polypeptides Promote the Survival of Glioma Cells

KLK6 polypeptides promoted the survival of human glioma cells. The U251human glioblastoma cell line was grown under control conditions or inthe presence of the protein kinase inhibitor staurosporine (1 μM), whichinduced a considerable increase in cell death. Cell death was measuredby flow cytometric detection of propidium iodide labeling. When gliomacells were pre-exposed to KLK6 polypeptides for 2 hours prior toapplication of staurosporine, a significant decrease in cell death wasobserved (FIG. 14; P<0.005, One Way ANOVA, SNK post hoc test).

In other assays, recombinant KLK6 was demonstrated to reduce death of aglioblastoma multiforme cell line, U251, in a dose dependent fashion inthe presence of the tyrosine kinase inhibitor staurosporine (FIG. 15).Annexin V labeling in combination with propidium iodide (PI) was used todemonstrate that recombinant KLK6 not only reduced the number of deadGBM cells, but also the number of apoptotic cells while simultaneouslyincreasing the number of live cells (FIG. 16).

In addition, KLK6 polypeptide over expression promoted the resistance ofU251 GBM cells to staurosporine induced cell death (FIG. 17) as well asto radiation (RT) and temozolomide (TMZ) alone, or in combination (FIGS.18 and 19). RT plus TMZ is a current standard of care for GBM patients,and targeting KLK6 may therefore sensitize these tumor cells toconventional therapies, thereby improving patient survival. Thesefindings were demonstrated using a KLK6 over expression construct andclonogenicity assays (FIGS. 18 and 19). Recombinant KLK6, or KLK6polypeptide over expression, also promoted resistance of U251 GBM cellsto the chemotherapeutic, cisplatin (FIG. 20). In GBM U251 cells, downregulation of PAR1 using a PAR1 targeting small hairpin vector blockedthe ability of KLK6 to promote resistance to cell death induced bystaurosporine. Thus, targeting PAR1 is another method to target theability of KLK6 to alter cell survival in GBM (FIG. 21).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-6. (canceled)
 7. A method for treating a mammal having cancer, whereinsaid method comprises administering a composition comprising aninhibitor of KLK6 polypeptide expression or activity to said mammalunder conditions wherein said composition increases the level ofapoptosis of cancer cells within said mammal.
 8. The method of claim 7,wherein said composition comprises a KLK6 antisense molecule or a KLK6miRNA molecule.
 9. (canceled)
 10. The method of claim 7, wherein saidmammal is a human.